Umbilical cord derived mesenchymal stem cell-GelMA microspheres for accelerated wound healing

Mesenchymal stem cells (MSCs) are an ideal seed cell for tissue engineering and stem cell transplantation. MSCs combined with biological scaffolds play an important role in promoting the repair of cutaneous wound. However, direct administration of MSCs is challenging for MSCs survival and integration into tissues. Providing MSCs with a biocompatible scaffold can improve MSCs survival, but the effect of gelatin methacrylate (GelMA) loaded MSCs from umbilical cord MSCs (UC-MSCs) in wound healing remains unknown. Here, we investigated the ability of GelMA with UC-MSCs complexes to promote migration and proliferation and the effect on wound healing in mouse models. We discovered that UC-MSCs attached to GelMA and promoted the proliferation and migration of fibroblasts. Both UC-MSCs and UC-MSCs-derived extracellular vesicles accelerated wound healing. MSC + Gelatin methacrylate microspheres (GMs) application decreased expression of transforming growth factor-β (TGF-β) and Type III collagen (Col3) in vivo, leading to new collagen deposition and angiogenesis, and accelerate wound healing and skin tissue regeneration. Taken together, these findings indicate MSC + GMs can promote wound healing by regulating wound healing-related factors in the paracrine. Therefore, our research proves that GelMA is an ideal scaffold for the top management of UC-MSCs in wound healing medical practice.


Introduction
Cutaneous wound repair is a restorative cellular response that includes hemostasis, tissue resolution, inflammation, cell proliferation, and tissue remodeling [1], and requires many cells and protein factors, such as fibroblasts [2], epithelium [3], basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factorβ (TGF-β), matrix metalloproteinase-3 (MMP3) and collagens [4,5]. Inference or miscoordination between cells and factors could lead to improper or impaired repair. Even normal wound healing in adult human skin can be delayed or result in scars [6]. For severely burned, diabetic, and senior populations, large wound areas, deep damage to skin, severe inflammation, and a lack of nutrients and oxygen make wound healing even more challenging, and more cells and factors are needed [7]. Cells, such as fibroblasts, endothelial progenitor cells, keratinocytes, MSCs, and platelets have been tested for wound healing in clinical practice [8].
MSCs are marked as CD70, CD90, and CD105 positive and CD34-negative cells [9]. MSCs can proliferate, regulate immune cells, secrete bioactive factors, and differentiate into adipocytes, osteoblasts, chondrocytes, and muscle cells in vitro [10]. MSCs widely exist in human tissues, such as synovium, dental pulp, bone marrow, ligaments, fat, skin, placenta, umbilical cord, and other fetal tissues, and have an integral role in native wound repair [11]. The bioactivity of MSCs from different tissues may vary. Hsieh et al reported that MSCs from umbilical cord MSCs (UC-MSCs) expressed more genes involved in angiogenesis and promoted microvasculature formation and cell migration better than MSCs from bone marrow [12]. MSCs have been extensively researched for medical use in wound healing, diabetes, stroke, autoimmune disease, neuronal degeneration, and many other diseases due to their differentiation potential, ease of harvest and expansion, and low immunogenicity in hundreds of clinical trials listed on the NIH [10]. MSCs show excellent safety in these clinical trials, barely cause any side effects. Evidence indicates that living MSCs could consistently modulate immune responses and promote cell migration, epithelialization, and agiogenesis to help wounds heal [13]. As a result, maintaining MSC bioactivity after delivery to injured cutaneous tissue is critical. However, direct administration of MSCs is challenging for MSCs survival and integration into tissues [14,15]. Therefore, insufficiency of MSCs engraftment at wounded areas is the major limitation for current MSC-based therapies.
Researchers developed several administration methods to improve MSCs survival, usually by providing MSCs with a biocompatible scaffold. The biomaterials used for fabricating the scaffold include natural or synthetic polymers and genetically engineered peptides [16,17]. Currently, many researchers are focused on MSC-laden hydrogels to treat skin wounds. For example, Farahnaz et al prepared a 3D collagen gel scaffold for placing MSCs on top [18]. The MSCs and scaffold complex promoted wound healing via early activation of MMPs and VEGF [19]. Montoya et al prove that a myogenic stem cell-laden hydrogel scaffold might be beneficial in the wound healing of the disrupted anal sphincter [20]. According to Ahmed et al, mesenchymal stem cell (MSC)laden, personalized 3D scaffolds with controlled structure and fiber alignment can promote granulation tissue formation, angiogenesis, and collagen deposition, as well as switch immune responses in the pro-regenerative direction [21]. Additionally, it has been shown that the application of BMSCs with NOreleasing hydrogel can promote faster regeneration in diabetic wounds [21]. Furthermore, new structures are being produced in order to offer adequate cell supports that could mimic their natural niche in a more realistic way. Previous results have shown that using gelatin microspheres as a carrier can improve the effects of platelet-rich plasma on the process of wound healing [22]. It has been demonstrated that the use of velvet antler polypeptide with PLGA microspheres enhanced the promoting effect of adiposederived mesenchymal stem cells (ADSCs) on wound surface repair by promoting the viability, proliferation, colony formation, and migration of ADSCs [23]. Gelatin methacrylate (GelMA) is a natural biomaterial derived from the hydrolytic degradation of collagen. GelMA is biosafe and commonly used in wound dressing applications. A variety of cell types, including fibroblasts [24], chondrocytes [25], endothelial cells, and human umbilical vein endothelial cells, have been used with GelMA [26]. However, GelMA has not been tested as a scaffold for UC-MSCs in wound healing.
In our study, we confirmed the biocompatibility of GelMA with UC-MSCs and generated MSC + Gelatin methacrylate microspheres (GM) complexes (figure 1). We found that MSC + GMs promote fibroblast migration and proliferation. Experiments on mice show that MSC + GMs are safe and can regulate inflammation factors, promote collagen synthesis, and re-epithelialize injured areas. In vitro and In vivo experiments suggest that MSC + GMs facilitate wound healing by paracrine to regulate factors involved in wound healing. Therefore, our studies prove that GelMA is an ideal scaffold for top administration of UC-MSCs in wound healing medical practice.

UC-MSC culture and expansion
Human umbilical cord Wharton's jelly samples were donated by women with full-term caesarean section and collected in Dongguan Peoples Hospital. All procedures involving human umbilical cords in this research were approved by the Ethical Review Board at Dongguan Peoples Hospital the research was conducted in accordance with the principles embodied in the Declaration of Helsinki and in accordance with local statutory requirements. Donators or their families were fully informed about this research and signed informed consent forms. Wharton's jelly samples were collected from healthy, full-term women and processed within 24 h of natural delivery. After the collection, the Wharton's jelly was transferred to the laboratory of the Dongguan Enlife Stem Cell Biotechnology Institute (Dongguan, China), washed in sterile phosphate buffered saline (PBS) (Hyclone, USA) three times to remove red blood cells, immersed in 70% ethanol for 30 s, and then immediately washed in PBS before further processing. Wharton's jelly was minced into small pieces (1-2mm 3 ) with a sterile scalpel, placed into 10cm 2 plates, and grown in media containing serum-free medium (Hyclone, USA), 5% UltraGROTM-Advanced (Helios Bioscience, USA). Then it was incubated in a humidified atmosphere containing 5%CO 2 at 37 • C. The medium was replaced every two days. The cells migrated from the explant to the medium margins. The adherent cells were harvested with 0.25% trypsin-ethylene diamine tetra acetic acid (EDTA) (Gibco, Germany) after the cells had reached 90% confluence in the second changing medium. The MSCs at the third passage were used for the experiments.

Osteogenic differentiation
UC-MSCs were cultured in six-well plates at an initial density of 4.5 × 10 5 cells/well and induced for osteogenic differentiation by culturing in the Osteogenic Differentiation Kit (Cyagen, China). Calcium mineralization was investigated through Alizarin Red S staining after 21 days of osteogenic induction.

Adipogenic differentiation
UC-MSCs were cultured in 6-well plates at an initial density of 4.5 × 10 5 cells/well and induced for adipogenic differentiation by culturing in the Adipogenic Differentiation Kit (Cyagen, China) according to the instructions. The lipid droplet formation exhibited by Oil Red O staining after 21 days of adipogenic induction.

Chondrogenic differentiation
UC-MSCs were cultured in 24-well plates at an initial density of 1 × 10 5 cells/well with a volume of 5 µl and induced in a humidified atmosphere of 5% CO2 at 37 • C for 3 h. Then the cells were induced to undergo chondrogenic differentiation by culturing in the Chondrogenic Differentiation Kit (Cyagen, China) according to the instructions. The cartilaginous structure of the pellets was verified by alcian blue staining after 21 days of chondrogenic induction.

Microspheres characterization
GelMA microspheres were coated with gold, and their morphology and particle size were examined using scanning electron microscopy (SEM; JSM-IT-100, Japan), with an accelerating voltage of 20 kV. Each sample was repeated 3 times.

Preparation of MSC + GM complexes
Place the tiled GelMA microspheres (EFL-MS-C-GM-100, Suzhou, China) without stacking under the ultraviolet lamp of the ultraclean platform for irradiation for 1-2 h. Transfer sterile microspheres into the spinner flask bioreactor. Add the culture medium to the spinner flask bioreactor to immerse the microspheres, place them in the 37 • C incubator for 30 min, and absorb and discard the culture medium after the microspheres are fully swollen and balanced. Add the cell suspension to the spinner flask bioreactor and culture overnight. 8-week-old male Swiss mice (body weight 18-20 g) were purchased from the Guangdong Medical Laboratory Animal Centre (Guangdong, China) and maintained under a 12 h light/dark cycle with unlimited access to food and water. All procedures were carried  Col3  CTGTAACATGGAAACTGGGGAAA  CCATAGCTGAACTGAAAACCACC  TGF-β1  CCACCTGCAAGACCATCGAC  CTGGCGAGCCTTAGTTTGGAC  TGF-β3  GGACTTCGGCCACATCAAGAA  TAGGGGACGTGGGTCATCAC  HIF-1  GAGGAAGGAGAAATCCCGTGA  TATGTGTCCGAAGGAAGCTGA  FGF1  GGGGAGATCACAACCTTCGC  GTCCCTTGTCCCATCCACG  MMP3 GGCCTGGAACAGTCTTGGC TGTCCATCGTTCATCATCGTCA β-Actin GTGACGTTGACATCCGTAAAGA GCCGGACTCATCGTACTCC out in accordance with the guidelines of the Guangdong Medical Experimental Animal Center's Animal Care and Use Committee and the Experimental Animal Ethics Committee. The mice (n = 20) were anesthetized by intraperitoneal injection of pentobarbital at 0.01 ml g −1 dose. Then the dorsal hair was shaved, and the skin was sterilized with 70% alcohol. Surgical scissors were used to create two full-thickness skin wounds (1.0 × 1.0 cm 2 ) on the right and left sides of the mouse's back, and penicillin-streptomycin was administered to prevent infection. Microspheres containing UC-MSCs or GelMA Microspheres were applied to the wounded area on the left, and 0.9% saline was applied to the wounded area on the right. To minimize wound skin contraction, which usually happened on the first day of the experiment, splinting rings (Grace Bio-Labs, USA) were centered on the wound area to fix the skin nylon sutures as previously shown [27,28]. The wound size was measured daily with a digital caliper. To test whether MSCs could differentiate and replace injured tissues, 100 µl microsphere combined with 1.0 × 10 7 ADSCs was inoculated subcutaneously on the back of male Swiss mice. The animals were sacrificed 15 days after the inoculation by cervical dislocation, and the subcutaneous adipose tissue from the backs of the mice was collected and weighed. Adipocytes were isolated from mouse adipose tissue by collagenase digestion for karyotyping and polymerase chain reaction (PCR) analyses.

Cell adhesion on microspheres
To culture the cells in suspension, cell culture dishes were coated with 0.9% agarose gel (Bioweste, Spain). 3 × 10 6 UC-MSCs and microspheres were mixed 1:1 in 500 µl of culture medium and incubated overnight in the precoated dishes. After 24 h of incubation, 500 µl fresh cell culture medium was added, and the microspheres were left undisturbed for 30 min. The unattached cells were washed away by sterile PBS, and the microspheres were stained with DAPI and observed under a fluorescence microscope (Zeiss, Germany) for the attached cells.

Cell counting kit 8 (CCK8) assay
The CCK-8 assay was used to evaluate cell proliferation. Cells were seeded onto 96-well plates (Thermo, USA) at a density of 5 × 10 4 cells/well in 100 µl of culture, then exposed to microspheres or MSC + GMs for 24, 48, and 72 h. The experiments were performed in triplicate. After incubation with GM or MSC + GMs, 10 µl CCK-8 solution (Dongjido, Japan) was added to each well and incubated for 4 h at 37 • C according to the manufacturer's instructions. The absorbance was measured at 450 nm using a Multiskan FC microplate photometer (BioTek, USA).

Total RNA extraction and quantitative real-time RT-PCR
Quickly transfer the skin tissue frozen in liquid nitrogen into a mortar precooled with liquid nitrogen. With liquid nitrogen, grind the tissue into a fine powder with a pestle and transfer the powder to an RNase-free 1.5 ml centrifuge tube precooled with liquid nitrogen. Total RNA from cells or tissue was extracted using TRIzol (Invitrogen, USA). The purity and concentration of RNAs were measured by absorbance on NanoDrop2000 (Thermo, USA) at 260 nm and 280 nm. RNA samples with OD260/OD280 between 1.8 and 2.0 were used to synthesize cDNA according to manufacturer's instructions (Transgen, China). Each real-time PCR was prepared in a 20 µl reaction mixture and performed on a CFX96 Touch Real-Time PCR Detection System (Biorad, USA). The real-time PCR reaction was performed with Green qPCR SuperMix (Transgen, China) and the thermocycling conditions were set according to the manufacturer's instructions. PCR primer (Invitrogen, USA) sequences for gene expression analyses were presented in table 1. Target Gene relative mRNA levels were calculated with normalization to β-actin values using the 2 −△△ct method.

Wound healing assay
Human dermal fibroblast (HDF) cells were seeded at 2 × 10 4 cells/well density in a two-well silicone insert with a defined cell-free gap (Ibidi, Germany) and grew in culture medium for 48 h. After the cells reached 100% confluence, the culture insert was removed, and the area that remained clear of cells was quantified by microscope (Zeiss, Germany) 24 h later. MSC + GMs were seeded at 2 × 10 4 cells/well into the upper chamber of 24-well transwell plates (Corning, USA) with 8 µm pore filters. Digital images were obtained at 0 h, 17 h, and 24 h.

Histology and immunochemical analysis
The animals were sacrificed on day 15, and the wound tissues were isolated from the back. For hematoxylin and eosin staining (H&E) and Masson's trichrome staining, skin tissue samples were fixed with 4% paraformaldehyde, the nuclei were stained with haematoxylin, and collagen was stained with a Masson's trichrome staining kit (Solarbio, China). About 4 µm thick paraffin embedded tissue sections were deparaffinized and heated in antigen retrieval buffer in a microwave for 30 min. For Ki67 immunohistochemistry staining, Ki67 primary antibody (Beyotime Biotechnology, China), HRP-conjugated anti-mouse IgG polymer (Beyotime Biotechnology, China), and DAB were used. Slides were weakly counterstained with hematoxylin diluted 1:5 in distilled water for 3 min, washed with tap water, and completely dried on a hot plate.

Chromosome analysis
For metaphase preparation, animals were killed by cervical dislocation. Cells were isolated from subcutaneous adipose tissues collected from the backs of 3 untreated or MSC + GMs treated mice (15 days after treatment). Cells were fixed in acetic acid and methanol (1:3) after 15 min in a hypotonic solution. Slides were prepared by the flame drying method and stained in 5% Giemsa for 5 min. Images of metaphase spreads were taken under a confocal microscope (Zeiss, Germany). For the karyotype study, the slides were coded randomly, and 100 well spread metaphase plates were studied from each mouse. 5 ′ -AAGTGATGCGAGTCCAGAAGG-3 ′ ; Human AP5M1 Forward: 5 ′ -AGATTCTCCAGACGG TATCCAA-3 ′ , Human AP5M1 Reverse: 5 ′ -CACG ACTCTCAACGAAGTCTTTA-3 ′ . DNA agarose gel electrophoresis was performed for 60 min at 80 V with 0.9% agarose gel in a buffer containing trisacetate EDTA (Ethylenediaminetetraacetic Acid) and 30 µl of DNA staining dye [29].

Statistical analysis
One-way analysis of variance and the t-test were used to analyze significant differences. Each experiment was performed in at least three independent cultures/animals per genotype or treatment condition, and data are presented as mean ± SEM. Statistical analyses were performed by GraphPad Prism 6 Software (GraphPad Software Inc., USA). The P value < 0.05 was considered as statistically significant.

In vitro attachment, proliferation capacity of MSCs on GelMA microspheres
Biomaterials are increasingly used for numerous medical applications, from the delivery of cancertargeted therapeutics to the treatment of cardiovascular diseases. The issue of foreign body reactions induced by biomaterials must be controlled to prevent treatment failure. Therefore, it is important to assess the biocompatibility and cytotoxicity of biomaterials in cell culture systems before proceeding to in vivo studies in animal models [30]. MSCs' survival and migration significantly affect the efficacy of cell transplantation, and a biocompatible scaffold would help. To determine the biocompatibility and applicability of GelMA microspheres as a scaffold for transplanted UC-MSCs, we prepared human UC-MSCs from fresh umbilical cord as described by Yi et al [31]. The characterization of passage 3-5 (P3-5) cells (supplementary figure S1) shows that UC-MSCs express stem cell specific markers and are highly bioactive. Under SEM analysis, the GelMA microspheres demonstrated a spherical shape, and the surface was found to be smooth (figure 2). GelMA microspheres were then mixed with UC-MSCs in suspension culture at a 1:1 ratio ( figure 3(A)). As shown in figure 3(B), after 24 h co-culture of GelMA microspheres and UC-MSCs (MSC + GMs) in a spinner flask bioreactor, UC-MSCs adhered to the microspheres. Live cell staining with DAPI showed that UC-MSCs-EGFP attached well to the surface of the microspheres. We further cultured the MSC + GMs in cell flasks for another 24 h and detected cell migration. Compared with the control cells, the presence of microspheres significantly improved UC-MSC survival through the non-adherent surface of EP tubes, as shown by increased cell density ( figure 3(A)). These data suggest that microspheres would help UC-MSC survive in a less friendly environment. To further evaluate the toxicity of GelMA to UC-MSCs, we co-cultured GelMA and MSCs and found GelMA slightly reduced cell viability, adhesion, and proliferation of UC-MSCs, compared with a non-GelMA environment (figures 3(C)-(E)). The addition of GelMA microspheres showed no significant differences in cell viability. The adhesion of UC-MSCs in the MSC + GMs group was slightly reduced at 8 h. The reason for this test result may be that with the continuous proliferation of MSCs, the surface area of microspheres available for MSCs adhesion decline, leading to the decrease of cell adhesion percentage. The addition of GelMA microspheres 48 h later reduce the proliferation of stem cells, indicating that the microspheres were slightly toxic to cells, which may be related to the degree of methylation of GelMA. These results suggest that GelMA microspheres only slightly affected the viability, adhesion and proliferation of stem cells over time.

Effects of MSC + GMs on HDFs in vitro
Fibroblasts are the major stromal cells in the dermis and play a critical role in the wound healing process by releasing numerous cytokines that promote wound healing [32]. UC-MSCs may help wound healing by stimulating fibroblast migration and proliferation. To test this hypothesis, we assessed the effect of MSC + GMs on HDFs cells' migration using wound healing assays. As shown in figure 4(A), after 24 h exposure to MSC + GMs, HDFs exhibited higher migration compared to untreated cells, indicating that MSC + GMs promote the motility of HDFs. However, HDFs have not shown wound healing properties in the GMs group compared to control cells. In addition, by using the CCK-8 cell viability assay kit, we found that MSC + GMs treated fibroblasts also increased cell viability compared with the control group. No significant differences were found between the GMs group and control group ( figure 4(B)).

The MSC + GMs improve wound healing in vivo
As MSC + GMs stimulate the migration potential of fibroblasts, it may contribute to the wound healing in vivo. Therefore, we evaluated the efficacy of MSC + GMs for wound healing in a mouse model by generating identical wounds on the left side and right side of a mouse back. As shown in figure 5(A), the sizes of wounds in MSC + GMs group were significantly smaller than control group and GMs group six days after treatment. On day 15, MSC + GMs treated wounds were fully healed, while the control group and GMs group had more than 25% unhealed areas ( figure 5(B)). Histologic and immunostaining analyses of the wounded areas demonstrated that MSC + GMs treatment generated thicker epithelium, more newly generated vesicles (figure 5(C)), more dermal collagen synthesis and regeneration ( figure 5(D)), and more Ki67-positive epithelial cells ( figure 5(E)). These findings suggest that MSC + GMs accelerate skin wound healing by increasing epithelial and dermal cell proliferation and dermal collagen synthesis in vivo.

Gene expression analysis of key wound healing proteins and inflammatory cytokines
Previous wound healing research have shown that the deposition of collagen and the expression of key growth factors, such as bFGF, VEGF and TGF-β, are crucial for tissue regeneration [33]. To investigate the molecular mechanisms of MSC + GMs in improving wound healing, we examined the mRNA expression of several fibroblast markers and inflammatory cytokines (collagen-I, collagen-III, TGF-β1, TGF-β3, FGF-1, HIF-1 and MMP3) on day 15 and found that MSC + GMs treatment significantly upregulated the expression of collagen-III, TGF-β1, TGF-β3, while the expression of MMP-3 was downregulated ( figure 6, supplementary figure S2). These data suggest that MSC + GMs accelerate wound healing by activating fibroblasts.

Therapeutic potentials of MSC + GMs
To explore the approach by which MSC + GMs accelerate wound closure, we hypothesized that     exosomes derived from MSCs might play a crucial role in promoting fibroblast migration and proliferation. We examined the effects of exosomes derived from UC-MSCs (supplementary figure S3) on the migration of fibroblasts in vitro (figure 7(A)) and found that the migration of fibroblasts was markedly increased when exposed to exosomes derived from MSCs ( figure 7(A)). MSCs exosomes facilitate fibroblast migration in a dose-dependent manner ( figure 7(A)). We also test whether MSCs could differentiate and replace injured tissues by evaluating the ability of the adipose derived mesenchymal stem cells in microspheres (AD-MSC + GMs) to form adipocytes in vivo. AD-MSC + GMs and AD-MSCs were implanted subcutaneously in mice and significantly increased subcutaneous adipose weights compared with the control group. In addition, AD-MSC + GMs increased more adipose weights than the AD-MSCs group ( figure 7(B)). Metaphase karyotyping of the adipocyte cells in the adipose tissue from mice injected with AD-MSC + GMs or AD-MSCs shows a mouse karyotype but not a human one ( figure 7(C)). In addition, PCR analysis did not detect the human adipocyte marker gene AP5M1 in adipose samples collected from mice ( figure 7(D)). These analyses eliminate the possibility that AD-MSCs transplanted into mice differentiate into adipocyte cells and indicate that MSCs on MSCs + GMs do not differentiate into damaged tissues to repair wound areas.
In this study, we found GelMA is compatible with UC-MSCs and could help UC-MSCs survive in harsh environments. The UC-MSC and GelMA complex aided mouse wound healing, most likely by promoting fibroblast migration and regulating wound healing factors, but not by directly replacing damaged tissues. The high efficacy and lack of integration into recipient tissues make MSC + GMs a promising and safe potential therapy for non-healing trauma.

Discussion
MSCs used as a medicine administered systemically or locally to cure hard-to-heal wounds is an intriguing possibility and is under clinical test [34]. However, no MSC based medicine has been approved yet. Systemic administration is friendly for MSCs' survival, and several studies have shown that MSCs are able to migrate to injuries in the host [11]. However, the long-term carcinogenic effects of systemically administered MSCs and the PK/PD for MSCs are difficult to determine due to the lack of tracking technology for MSCs in humans. On the other hand, local administration requires long-term retention of MSCs in the wound area. Therefore, several biocompatible materials designed as a niches for MSCs attachment were tested, mainly on traditional bone marrow MSCs [35], and a few studies investigated umbilical cord MSCs [36][37][38]. Despite being discovered later, UC-MSCs are easier to harvest and proliferate.
GelMA is a gelatin derivative and is biocompatible, cleavable to MMPs, friendly for cell adhesion, easy to prepare, and has tailorable mechanical properties. Therefore, GelMA hydrogels have been widely used for various biomedical applications, including tissue engineering, drug delivery, and 3D printing [39,40]. Xu et al reported that GelMA microspheres can sustain bone marrow MSCs' viability, support cell spreading inside the microspheres, and even enhance MSCs' proliferation [41]. Donnell et al reported that adipose MSCs retain adipogenic potential when seeded within GelMA hydrogels [42]. Zhang et al successfully cocultured umbilical cord MSCs with GelMA [43], indicating GelMA might be biocompatible with MSCs. In this research, we generated GelMA microspheres with a diameter of 100 µm and co-cultured microspheres with UC-MSCs. GelMA shows great biocompatibility, but UC-MSCs co-cultured with GelMA grow slightly slower than UC-MSCs grown directly on a cell culture dish. As the hydrolysis product of collagen, GelMA contains many arginine-glycine-aspartic acid sequences that promote cell attachment [44] and can serve as a niche for MSCs to grow, though likely not as well as cell culture dishes, because UC-MSCs co-cultured with GelMA microspheres on a cell culture dish grow less well than dishes alone.
In the wounded area, fibroblasts migrate to the wound matrix and release collagen, fibronectin, and proteoglycans, then replace the initial fibrin matrix with new extracellular matrix. Our data indicate that MSCs that attach to GelMA promote wound healing by enhancing fibroblast migration and vesiculation and promoting protein and gene expression. Type III collagen, TGF-1, and TGF-3 genes are found to be up-regulated, while MMP3 is down-regulated in tissues treated with MSC + GMs. Type III collagen is a major component of the extracellular matrix in skin and many internal organs [45]. TGF-1 and TGF-3 are TGF-family members that play critical roles in wound healing, including inflammation control, angiogenesis, fibroblast proliferation, collagen synthesis and deposition, and extracellular matrix remolding [28]. MSC + GMs help wound healing by promoting growth factor secretion and regulating immune microenvironment in the wounded tissues.

Conclusion
MSCs administered into the body may directly differentiate into daughter cells and form building blocks of tissue renewal (such as keratinocytes, fibroblasts, and pericytes); modulate immune cells; secrete growth factors required for wound healing by paracrine signaling; and mobilize the resident stem cell niche to promote wound healing. In this study, we found that administering MSCs caused excessive adipose tissue accumulation in mice, despite the fact that none of the adipocytes were from humans. This phenotype indicates that the tissue regeneration is likely not from direct differentiation of MSCs but rather through paracrine means. In the future, further investigation about the molecular mechanism of how MSC + GMs regulate factor expression, migrate fibroblasts, regulate the immune environment, promote the epithelium, and promote vascular growth is needed. The necessary development of technical parameters of MSC + GMs complexes to improve efficacy is also required to develop a new medicine for hard-to-heal wound indications.

Data availability statement
The datasets generated and/or analyzed during the current study are not publicly available due but are available from the corresponding author on reasonable request.
All data that support the findings of this study are included within the article (and any supplementary files).